Human OPRM1 and murine Oprm1 promoter driven viral constructs for genetic access to μ-opioidergic cell types

With concurrent global epidemics of chronic pain and opioid use disorders, there is a critical need to identify, target and manipulate specific cell populations expressing the mu-opioid receptor (MOR). However, available tools and transgenic models for gaining long-term genetic access to MOR+ neural cell types and circuits involved in modulating pain, analgesia and addiction across species are limited. To address this, we developed a catalog of MOR promoter (MORp) based constructs packaged into adeno-associated viral vectors that drive transgene expression in MOR+ cells. MORp constructs designed from promoter regions upstream of the mouse Oprm1 gene (mMORp) were validated for transduction efficiency and selectivity in endogenous MOR+ neurons in the brain, spinal cord, and periphery of mice, with additional studies revealing robust expression in rats, shrews, and human induced pluripotent stem cell (iPSC)-derived nociceptors. The use of mMORp for in vivo fiber photometry, behavioral chemogenetics, and intersectional genetic strategies is also demonstrated. Lastly, a human designed MORp (hMORp) efficiently transduced macaque cortical OPRM1+ cells. Together, our MORp toolkit provides researchers cell type specific genetic access to target and functionally manipulate mu-opioidergic neurons across a range of vertebrate species and translational models for pain, addiction, and neuropsychiatric disorders.


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Randomization
All source and raw data generated in this study have been deposited in the Zenodo database under the accession code ().Additional requests for further data or information regarding available data files will be addressed upon request to the corresponding authors.
No statistics were used to determine sample sizes.Sample sizes for chemogenetic and fiber photometry testing were chosen based on common practices that have historically been reported within the murine behavioral research field, including instances from previously peer reviewed publications authored by the lead and communicating authors (see PMID: 30655440, PMID: 32277042, PMID: 32074627 and PMID: 36941364 for examples).Standard practices reported for average sample sizes deemed sufficient to allow for well powered statistical analyses of both in situ and immunohistochemical studies for examining transcript or protein expression profiles across animals were also applied as previously reported in the literature (see PMID: 25600267 and PMID: 32277042 for previous, peer-reviewed examples).Sample sizes were not taken into account for experiments in which the end goal was qualitative, not quantitative, and primarily based on the ability to determine successful and efficient transduction of neural tissue via one of our viruses.In these instances, as reported in the manuscript text and additional reporting documents, cohort sizes for each set of injections with these viruses were set at a high enough number to hopefully allow for the successful transduction of target sites in at least one animal (groups typically consisted of 5+ animals per virus and per set of injections).As previous work in our lab in done to test or injection animals with new or untested virus had shown that at least 2-3 animals would be sufficient to account for any technical error that could occur over the course of a single stereotaxic surgery, a slight larger number of animals for this testing was deemed sufficient.
For all imaging and behavioral studies, virus injected animals with either little or no evidence of viral transduction and/or incorrect viral targeting were excluded from any final analyses.Poor or loss of coupling between the fiber optic patch cord and the fiber optic implant on mice during fiber photometry testing led to the data collected from that animal for a given test to be discarded.No other mice or data points were excluded across analyses.
Virus transduction efficiency, specificity and restriction patterns were reliably reproduced across multiple cohorts of animals from experimental species discussed in the text (where applicable).Experimental findings in imaging studies regarding viral specificity were found to be reliably replicated across multiple cohorts of animals.In behavioral testing, findings within experimental and control groups were also found to be readily replicated.As multiple cohorts were not used for such studies, steps were taken to ensure that group sizes for all behavioral studies were sufficiently powered, as per standard animal behavioral research practices.Indication of data from individual subjects across all tests are displayed in figures where applicable.Detailed information and protocol outlines are additionally provided in figure legends and the Methods section, respectively, to ensure reproducibility.
For spinal chemogenetic behavioral experiments, the order of group testing was randomized, but not blinded, such that we alternated testing mMORp-hM4Di-mCherry and then hSyn-mCherry control mice to control for testing order and time of day.For in vivo fiber photometry

Blinding
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Antibodies
Antibodies used testing, group testing was not randomized, with experimental and control virus injected animal groups run consecutively over the course of the testing day, with the experimental group run first.Randomization and blinding were not deemed possible for these studies, as the simultaneous calcium event recording that was conducted during behavioral testing would have immediately identified GCaMP6f+ mice and eYFP+ control mice due to the presence of detectable signal for GCaMP activity in the experimental group mice and the complete lack of this signal in control group mice (as demonstrated by the relevant data and figures provided for these studies in our manuscript).As such, animals were run as separate experimental and control groups for these studies, with photometry and behavioral data scored after initial acquisition.Following this logic, chronic morphine drinking and saccharin drinking groups were run consecutively and as block groups as well during fiber photometry testing.For all other histological and viral validation based experiments discussed in this manuscript, randomization was also not applied, as it for the purposes of either of these sets of quantitative or qualitative analyses, random allocation across experimental groups was not deemed necessary.As the end goal of our quantitative histological studies was specifically to determine if our viral tools did indeed more selectively transduce MOR/Oprm1/OPRM1+ cells more selectively over other cell types within the same tissue sample, group allocation was not necessary (nor randomization needed) as all animals used in these studies were injected with the same experimental viruses.Similarly, for viral validation studies, as the end results of these experiments was deemed to be observation of either successful viral transduction or not, knowledge of the injected viruses was already known, and confirmation of expression (or lack there was) was deemed to be a fairly definitive outcome, and not amenable (or necessary) to be subject to randomization.
For chemogenetic behavioral testing, data were analyzed by a second experimenter, blinded to group identification.Order and identity of experimental or control virus injected groups were not blinded for initial in vivo fiber photometry testings or data analysis as the identity of these groups was given away with the presence of measurable calcium mediated events for initial testing.These initial tests also served primarily to explore the functionality of this viral tool more than anything as well.Built upon this same basis, experimenters were not blinded to group identification for acute morphine treatment or chronic morphine drinking assays.For all other histological and viral validation testing designed to assess both viral specificity and overall transduction efficiency, blinding was deemed as not possible or unnecessary across groups as the end goal of these studies was either to quantify the overall transduction specificity of an already known viral species or to simply validate the successful or unsuccessful expression of a viral species at injections sites across different neural structures.Even in the case where different viruses were co-injected for select specificity assays, specific fluorophores associated with and necessary to identify which virus was able to transduce which cells, made the identity of each viral species used difficult to blind experimenters to, and somewhat unnecessary, as it was crucial to be able to distinguish one type of virus from another for the successful interpretation and quantification of the results for these studies in general.As no specific manipulations were performed on the animal cohorts that compromised each of the viral injection groups across all histological assays (both IHC and ISH based), no additional blinding was deemed necessary for the effective analysis of these experiments.